The insulin-like growth factor I receptor called IGF-IR is a receptor with tyrosine kinase activity having 70% homology with the insulin receptor IR. IGF-IR is a glycoprotein of molecular weight approximately 350,000. It is a hetero-tetrameric receptor of which each half—linked by disulfide bridges—is composed of an extracellular α-subunit and of a transmembrane β-subunit IGF-IR binds IGF1 and IGF2 with a very high affinity (Kd #1 nM) but is equally capable of binding to insulin with an affinity 100 to 1000 times less. Conversely, the IR binds insulin with a very high affinity although the IGFs only bind to the insulin receptor with a 100 times lower affinity. The tyrosine kinase domain of IGF-IR and of IR has a very high sequence homology although the zones of weaker homology respectively concern the cysteine-rich region situated on the α-subunit and the C-terminal part of the β-subunit. The sequence differences observed in the α-subunit are situated in the binding zone of the ligands and are therefore at the origin of the relative affinities of IGF-IR and of IR for the IGFs and insulin respectively. The differences in the C-terminal part of the O-subunit result in a divergence in the signalling pathways of the two receptors; IGF-IR mediating mitogenic, differentiation and antiapoptosis effects, while the activation of the IR principally involves effects at the level of the metabolic pathways (Baserga et al., Biochim. Biophys. Acta, 1332:F105-126, 1997; Baserga R., Exp. Cell. Res., 253:1-6, 1999).
The cytoplasmic tyrosine kinase proteins are activated by the binding of the ligand to the extracellular domain of the receptor. The activation of the kinases in its turn involves the stimulation of different intra-cellular substrates, including IRS-1, IRS-2, Shc and Grb 10 (Peruzi F. et al., J. Cancer Res. Clin. Oncol., 125:166-173, 1999). The two major substrates of IGF-IR are IRS and Shc which mediate, by the activation of numerous effectors downstream, the majority of the growth and differentiation effects connected with the attachment of the IGFs to this receptor. The availability of substrates can consequently dictate the final biological effect connected with the activation of the IGF-IR. When IRS-1 predominates, the cells tend to proliferate and to transform. When Shc dominates, the cells tend to differentiate (Valentinis B. et al., J. Biol. Chem. 274:12423-12430, 1999). It seems that the route principally involved for the effects of protection against apoptosis is the phosphatidyl-inositol 3-kinases (Pt 3-kinases) route (Prisco M. et al, Horm. Metab. Res., 31:80-89, 1999; Peruzzi F. et al., J. Cancer Res. Clin. Oncol., 125:166-173, 1999).
The role of the IGF system in carcinogenesis has become the subject of intensive research in the last ten years. This interest followed the discovery of the fact that in addition to its mitogenic and antiapoptosis properties, IGF-IR seems to be required for the establishment and the maintenance of a transformed phenotype. In fact, it has been well established that an overexpression or a constitutive activation of IGF-IR leads, in a great variety of cells, to a growth of the cells independent of the support in media devoid of fetal calf serum, and to the formation of tumors in nude mice. This in itself is not a unique property since a great variety of products of overexpressed genes can transform cells, including a good number of receptors of growth factors. However, the crucial discovery which has clearly demonstrated the major role played by IGF-IR in the transformation has been the demonstration that the R-cells, in which the gene coding for IGF-IR has been inactivated, are totally refractory to transformation by different agents which are usually capable of transforming the cells, such as the E5 protein of bovine papilloma virus, an overexpression of EGFR or of PDGFR, the T antigen of SV 40, activated ras or the combination of these two last factors (Sell C. et al., Proc. Natl. Acad. Sci., USA, 90:11217-11221, 1993; Sell C. et al., Mol. Cell. Biol., 14:3604-3612, 1994; Morrione A. J., Virol., 69:5300-5303, 1995; Coppola D. et al., Mol. Cell. Biol., 14:4588-4595, 1994; DeAngelis T et al., J. Cell. Physiol., 164:214-221, 1995).
IGF-IR is expressed in a great variety of tumors and of tumor lines and the IGFs amplify the tumor growth via their attachment to IGF-IR. Other arguments in favor of the role of IGF-IR in carcinogenesis come from studies using murine monoclonal antibodies directed against the receptor or using negative dominants of IGF-IR Actually, murine monoclonal antibodies directed against IGF-IR inhibit the proliferation of numerous cell lines in culture and the growth of tumor cells in vivo (Arteaga C. et al., Cancer Res., 49:6237-6241, 1989; Li et al., Biochem. Biophys. Res. Com., 196:92-98, 1993; Zia F. et al, J. Cell. Biol., 24:269-275, 1996; Scotlandi K et al., Cancer Res., 58:4127-4131, 1998). It has likewise been shown in the works of Jiang et al. (Oncogene, 18:6071-6077, 1999) that a negative dominant of IGF-IR is capable of inhibiting tumor proliferation.
Such antibodies capable to bind specifically to the IGF-IR have been described and several patent applications have been filed. As an example, we can mentioned the patent application WO 03/059951, filed by the Applicant, wherein a monoclonal antibody able to bind to IGF-IR, called 7C10, is described. Others patent applications can be mentioned as WO 02/053596 (PFIZER INC. and ABGENIX INC.), WO 03/100008 (SCHERING CORPORATION) or WO 03/106621 (IMMUNOGEN INC.). All these applications are claiming antibodies capable to specifically bind to the IGF-IR and/or to inhibit its activity.
Although it is considered as an evident property for each of these antibodies, nothing in the specification of these applications clearly demonstrate that these antibodies are capable to inhibit efficiently the binding of both natural ligands to IGF-IR and, more particularly the binding IGF2 to IGF-IR. In vitro data such as inhibition of proliferation IGF1 and IGF2 induced were shown in these applications and demonstrated that the described antibodies are able to inhibit efficiently both IGF1 and IGF2-induced proliferation. However, as the major part of described antibodies displayed the capacity of inducing a partial down-regulation of IGF-IR, it is not evident that the antiproliferative properties are directly linked to a displacement of both IGF1 and IGF2 from IGF-IR.
Actually, for example, data shown in WO 03/106621 are only dealing with IGF1 and under no circumstances with an eventual inhibition of the binding of the IGF2 to IGF-IR (see Example C, pages 33-35 and FIG. 3, and Example D, pages 35-37 and FIGS. 4-6).
The same observations can be made for WO 02/053596 (see Example TV, page 78-79 and FIG. 3, and Example VII, page 82 and FIG. 4).
Amongst all the presently identified patent applications describing the discovery of monoclonal or recombinant antibodies directed against the human IGF-IR (hIGF-IR), only two of them (WO 2004/087756 and WO 2005/005635) are really showing an efficacy of the antibodies, respectively AK1a and AK18, to inhibit [125I]IGF2 binding to hIGF-IR.
The experimental procedure, identical in both cases, is based on competition binding on human colorectal adenocarcinoma (HT29) intact cells. Although the described procedure is sound, no positive controls such as competition by natural hIGF-IR ligands (IGF1 and IGF2) is presented, making the data on competition binding suspicious. On the other hand, only incomplete competition was shown (maximally 80% inhibition of [125I]IGF2 binding for both AK1a and AK18 antibody), even at high antibody concentration, making the development of more efficacious antibodies necessary for enhanced therapeutic efficacy in humans. A last controversial point concerning the putative inhibition of IGF2-mediated responses by AK1a and AK18 is the absence of examples showing an inhibition of functional IGF2-stimulated signaling mediated by hIGF-IR. Indeed, even if competition of IGF2 binding is happening, it must be associated with a concomitant inhibition of downstream hIGF-IR signalling and such evidence must be exemplified to provide mechanistic evidence for antibody functional efficacy.